CN1910284A

CN1910284A - Epitope/peptide recognized by HLA-A2402-restricted Ep-CAM-specific CTL and use of the same

Info

Publication number: CN1910284A
Application number: CNA2005800028078A
Authority: CN
Inventors: 葛岛清隆
Original assignee: Ai Zhixian; Otsuka Pharmaceutical Co Ltd
Current assignee: Ai Zhixian; Otsuka Pharmaceutical Co Ltd; Aichi Prefecture
Priority date: 2004-01-20
Filing date: 2005-01-19
Publication date: 2007-02-07
Anticipated expiration: 2025-01-19
Also published as: US7619058B2; US20100167321A1; ES2390967T3; JPWO2005068632A1; EP1715042B1; WO2005068632A8; EP1715042A4; WO2005068632A1; CA2554001A1; EP1715042A1; CN101434647A; JP4779067B2; US7846651B2; US20090010951A1; KR20060129393A; CN1910284B

Abstract

A peptide consisting essentially of the amino acid sequence represented by SEQ ID NO:1; a peptide consisting essentially of the amino acid sequence represented by SEQ ID NO:2; or a mutant peptide consisting essentially of an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO:1 or 2 by addition, deletion or substitution of one or more amino acids, the peptide being capable of forming a complex with an HLA-A2402 molecule to be recognized by HLA-A2402-restricted cytotoxic T lymphocytes or induce such lymphocytes. Such a peptide is useful as a cancer vaccine for epithelial cancer patients having HLA-A2402.

Description

Epi-position/peptide and application thereof by the special CTL identification of the Ep-CAM-of HLA-A2402-restriction

Technical field

The present invention relates to epitope peptide, antigen presenting cell and major histocompatibility antigen complex body by born of the same parents' poison T lymphocyte identification of HLA-A2402-restriction; Contain cancer vaccine or the born of the same parents poison T lymphocyte inductor of these materials as activeconstituents; Contain the passive immunotherapy medicine of this kind born of the same parents poison T lymphocyte inductor as the epidermal carcinoma of activeconstituents; Utilize the cancer therapy or the improvement method of above-mentioned substance; And the lymphocytic quantivative approach of born of the same parents poison T that is specific to the HLA-A2402-restriction of cancer.

Background technology

Born of the same parents' poison T lymphocytes (hereinafter referred to as " CTL ") are considered to resist the important factor of cancer.

Tumor locus the cancer patients can be observed the CTL infiltration that shows cellular toxicity at tumour cell.Tumour antigen is by the molecule of this TS CTL target, in cell, resolve into and contain 8-11 amino acid whose peptide (tumour epitope peptide), be attached to human leucocyte antigen (HLA) (hereinafter referred to as " HLA " is a kind of major histocompatibility antigen), present at tumor cell surface then.CTL identification contains the complex body of HLA and tumor antigen peptide, and attacks tumour cell.Thus, CTL is by the HLA restriction tumor cell of tumour antigen.

The membrane antigen of HLA on nearly all cell, expressing, and mainly be divided into I type antigen and II type antigen.The HLA that is discerned by CTL with epitope peptide is divided into I type antigen.HLAI type antigen further is divided into HLA-A, HLA-B, and HLA-C etc., and also their gene also has hypotype.For example, HLA-A is a polymorphism, and has hypotypes such as A1, A2, A24, A26.Therefore, the HLA of each individual human is always not identical, and CTL is when the complex body of identification HLAI type antigen and tumour epitope peptide even can discern these HLA hypotypes.Further, can be attached to that the epitope peptide of HLA is known to have a peptide sequence primitive (mode sequences) that can be attached to one of various HLA.Therefore, for induce and/activation CTL, the peptide of selecting to contain the primitive that can be attached to each patient's different shaped HLA is necessary.

Ep-CAM is the molecule of wide expression on the cancer cells surface of epithelium genesis, and takes on the intermediate of non-Ca-dependent cell-cell attachment.Ep-CAM is also referred to as EGP-2,17-1A, GA733-2 or KSA.Ep-CAM is derived from such as large intestine, lung, head and neck are highly expression in many tumours of different tissues such as mammary gland origin, and its to be expressed in the normal epithelium cell be zonal.Further, because tumor development is relevant with the Ep-CAM expression level, so the serve as a mark metastases of the minimum existence of diagnosis and in predicting patient's prognosis, be useful of the detection expressed of Ep-CAM.

Ep-CAM is wide expression in being derived from epithelial cancer cells, but in normal cell local expression.Given this, Ep-CAM utilizes monoclonal antibody to carry out important target in immunotherapy and the gene therapy.

For example, report is arranged, give its tumour the patient by surgical removal use the special mouse monoclonal antibody of Ep-CAM-(called after 17-1A) and can effectively prevent remote transfer, and administration in continuous 7 years is effective to improving survival rate.Also have report, the special monoclonal antibody of the Ep-CAM-of called after 17-1A is effective to reducing mortality ratio and tumor recurrence when being used for the treatment of PATIENTS WITH LARGE BOWEL.

In addition, the nearest disclosed true Ep-CAM that utilizes CTL target HLA-restriction that shows of some reports treats the possibility of cancer.

For example, patent documentation 1 report, the Ep-CAM-specific CTL of HLA-A0201-restriction destroys epithelial tumor cell, but does not influence normal cell.Disclosed peptide is made up of the sequence YQLDPKFITSI shown in the SEQID NO:12 in the patent documentation 1, as the Ep-CAM 174-184 epitope peptide of the CTL identification that is limited by HLA-A0201-.

The T-cell is also observed in the colon of not accepting immunotherapy and PATIENTS WITH LARGE BOWEL the reaction of Ep-CAM.In addition, when the reorganization canary pox virus that the PATIENTS WITH LARGE BOWEL immunity is expressed with Ep-CAM, the acquisition of anti-Ep-CAM-specific CTL reaction can not cause autoimmune response.

In the antigenic HLA-A type of HLA I type, Japanese HLA-A24 expresses at most.As a result, the Ep-CAM epitope peptide of HLA-A24-restriction infers it is the ideal cancer vaccine.The report example of relevant this epitope peptide is as follows.

Patent documentation 2 discloses 5 kinds of epitope peptides, is made up of the aminoacid sequence that contains 9 amino-acid residues, from the SART-2 tumor antigen protein, as the CTL epitope peptide of HLA-A2402-restriction.

Patent documentation 3 discloses 6 kinds of epitope peptides, is made up of the aminoacid sequence that contains 8-11 amino-acid residue, from the ART-4 tumor antigen protein, as the CTL epitope peptide of HLA-A2402-restriction.

Patent documentation 4 discloses 7 kinds of epitope peptides, form by the aminoacid sequence that contains 8-11 amino-acid residue, come the p56luk albumen (by the tumor antigen protein of luk genes encoding) of unconventionality expression in comfortable colorectal cancer cells and the small cell lung cancer cell, as CTL epitope peptide available from the HLA-A2402-restriction of the clone of setting up from the esophagus cancer patient.

Patent documentation 5 discloses 4 kinds of PI-9-deutero-epitope peptides, form by the aminoacid sequence that contains 9-10 amino-acid residue, available from KE4 tumor cell line-deutero-cDNA library, as CTL epitope peptide available from the HLA-A2402-restriction of the clone of setting up from the esophagus cancer patient.

Patent documentation 6 discloses 17 kinds of PI-9-deutero-epitope peptides, form by the aminoacid sequence that contains 9-10 amino-acid residue, available from the 11-18 lung adenocarcinoma cell is the cDNA library, as the CTL epitope peptide available from the HLA-A2402-restriction of the clone of setting up from patients with lung cancer.

The patent documentation 7 open epitope peptides of forming by 9-10 amino-acid residue, has the primitive (HLA-A2402) that can be attached to HLA-A24.1, and has the first conserved residues Y at N-end, F or W, and has the second conserved residues F at C-end, I, W or M, wherein first and second conserved residues are by 6-7 residue separately.

As above institute's illustration, the CTL epi-position of the different HLA-A2402-restrictions in the tumor associated antigen is identified; Yet the expression of tumour antigen is according to the tissue origin, and individual cancer patient and each patient damage and change, so other new epi-positions are estimated designated.

In addition, owing to the CTL that derives by the Ep-CAM epitope peptide that can be attached to the HLA-A2402 molecule is still unconfirmed, therefore can be attached to the HLA-A2402 molecule and can induce novel cancer cells-special Ep-CAM epitope peptide of CTL to have demand exploitation.

Patent documentation 1:

International patent publications No.WO 97/15597

Patent documentation 2:

Do not examine Japan patent applicant announce No.1999-318455

Patent documentation 3:

Do not examine Japan patent applicant announce No.2000-116383

Patent documentation 4:

International patent publications No.WO2000-06595

Patent documentation 5:

Do not examine Japan patent applicant announce No.2001-245675

Patent documentation 6:

Do not examine Japan patent applicant announce No.2003-270

Patent documentation 7:

Do not examine Japan patent applicant announce No.1996-500103 (claim 11 etc.)

Summary of the invention

The problem that invention solves

The object of the invention provides the lymphocytic Ep-CAM epitope peptide of born of the same parents poison T that can induce the HLA-A2402-restriction, antigen presenting cell and major histocompatibility antigen complex body; Contain cancer vaccine or the born of the same parents poison T lymphocyte inductor of these materials as activeconstituents; Born of the same parents' poison T lymphocyte of Ep-CAM-specificity HLA-A2402-restriction; Contain the lymphocytic passive immunotherapy medicine of this kind; Utilize the cancer therapy or the improvement method of above-mentioned substance; And the lymphocytic quantivative approach of born of the same parents poison T of the special HLA-A2402-of epithelial cancer restriction.

The method of dealing with problems

To achieve these goals, the inventor predicts in Ep-CAM albumen by the information biology approach and has synthesized 7 kinds of peptide sequences, found and had the epitope peptide sequence that is attached to HLA-A2402 molecule ability, the HLA-A2402 molecule is modal HLA-A type (more than 60%) in the Japanese, and in the European about 20%.

In the time can being attached to the peptide of specificity HLA molecule by biological information approach prediction, by born of the same parents' poison T lymphocyte of the inducing peptide of this prediction in fact usually nonrecognition contain the antigen of this peptide.

In this case, the inventor finds, is specific to 2 kinds ctl clone in the epitope peptide of 7 kinds of predictions and really the positive Ep-CAM of HLA-A2402 expressed cancer cells and show cellular toxicity, but the negative cancer cells of HLA-A2402 is not shown cellular toxicity.

And cold target inhibition analysis proves that these epitope peptides are processed, and as antigen presentation on the positive Ep-CAM positive cancer cell of HLA-A2402.

As a result, these two kinds of peptides can be used as the cancer vaccine with HLA-A2402 people.

The present invention is based on above-mentioned discovery and finish, and following epitope peptide etc. is provided.

The 1st. each peptide of following (1)-(4):

(1) peptide of forming by the aminoacid sequence shown in the SEQ ID NO:1 basically;

(2) peptide of forming by the aminoacid sequence shown in the SEQ ID NO:2 basically;

(3) basically by passing through interpolation, disappearance or replace one or more amino acid and the mutant peptide formed derived from the aminoacid sequence of aminoacid sequence shown in the SEQ ID NO:1, described peptide can form complex body with the HLA-A2402 molecule and born of the same parents' poison T lymphocyte identification of being limited by HLA-A2402-or induce this lymphocyte;

(4) basically by passing through interpolation, disappearance or replace one or more amino acid and the mutant peptide formed derived from the aminoacid sequence of aminoacid sequence shown in the SEQ ID NO:2, described peptide can form complex body with the HLA-A2402 molecule and born of the same parents' poison T lymphocyte identification of being limited by HLA-A2402-or induce this lymphocyte.

The 2nd. the peptide of following (1) or (2):

(1) peptide of forming by aminoacid sequence shown in the SEQ ID NO:1 basically;

(2) peptide of forming by aminoacid sequence shown in the SEQ ID NO:2 basically.

The 3rd. the peptide that comprises the 1st or 2 is as the cancer vaccine of activeconstituents.

The 4th. the 3rd cancer vaccine, wherein cancer is an epithelial cancer.

The 5th. the 3rd or 4 cancer vaccine, wherein cancer is selected from large bowel cancer, lung cancer, mammary cancer, cancer of the stomach, oral carcinoma, carcinoma of the pancreas, esophagus cancer, nasopharyngeal carcinoma, uterus carcinoma, prostate cancer, and carcinoma of gallbladder.

The 6th. each cancer vaccine of 3-5, it is used for the treatment of and has the people of HLA-A2402 as human leucocyte antigen.

The 7th. the peptide that comprises the 1st or 2 is as the born of the same parents of activeconstituents poison T lymphocyte inductor.

The 8th. the 7th born of the same parents poison T lymphocyte inductor, it is used for the treatment of and has the people of HLA-A2402 as human leucocyte antigen.

The 9th. each polynucleotide of following (5)-(8):

(5) polynucleotide of forming by base sequence shown in the SEQ ID NO:10 basically;

(6) polynucleotide of forming by base sequence shown in the SEQ ID NO:11 basically;

(7) under stringent condition with the sudden change polynucleotide of the multi-nucleotide hybrid of forming by base sequence shown in the SEQ ID NO:10, the peptide of its coding can form complex body and be discerned or induce this lymphocyte by born of the same parents' poison T lymphocyte that HLA-A2402-limits with the HLA-A2402 molecule;

(8) under stringent condition with the sudden change polynucleotide of the multi-nucleotide hybrid of forming by base sequence shown in the SEQ ID NO:11, the peptide of its coding can form complex body and be discerned or induce this lymphocyte by born of the same parents' poison T lymphocyte that HLA-A2402-limits with the HLA-A2402 molecule.

The 10th. epitheliomatous gene therapy medicament, the polynucleotide that comprise the 9th are as activeconstituents.

The 11st. recombinant vectors comprises the 9th polynucleotide.

The 12nd. transformant wherein imports the 11st recombinant vectors.

The 13rd. the preparation method of the 1st or 2 peptide, comprise the transformant of cultivating the 12nd, and the step of from culture, collecting the 1st or 2 peptide.

The 14th. antigen presenting cell, its pulse is with the 1st or 2 peptide.

The 15th. cancer vaccine, the antigen presenting cell that comprises the 14th is as activeconstituents.

The 16th. the 15th cancer vaccine, wherein cancer is an epithelial cancer.

The 17th. the 15th or 16 cancer vaccine, wherein cancer is selected from large bowel cancer, lung cancer, mammary cancer, cancer of the stomach, oral carcinoma, carcinoma of the pancreas, esophagus cancer, nasopharyngeal carcinoma, uterus carcinoma, prostate cancer, and carcinoma of gallbladder.

The 18th. each cancer vaccine of 15-17, it is used for the treatment of and has the people of HLA-A2402 as human leucocyte antigen.

The 19th. born of the same parents' poison T lymphocyte inductor, the antigen presenting cell that comprises the 14th is as activeconstituents.

The 20th. the 19th born of the same parents poison T lymphocyte inductor, it is used for the treatment of and has the people of HLA-A2402 as human leucocyte antigen.

The 21st. the major histocompatibility antigen complex body, comprise major histocompatibility antigen and the 1st or 2 's peptide, perhaps be present in the tumour antigen epitope peptide on the 14th the antigen presenting cell.

The 22nd. the 21st major histocompatibility antigen complex body, comprise the HLA-A2402 molecule, B2M and the 1st or 2 's peptide perhaps is present in the tumour antigen epitope peptide on the 14th the antigen presenting cell.

The 23rd. cancer vaccine, the major histocompatibility antigen complex body that comprises the 21st or 22 is as activeconstituents.

The 24th. the 23rd cancer vaccine, wherein cancer is an epithelial cancer.

The 25th. the 23rd or 24 cancer vaccine, wherein cancer is selected from large bowel cancer, lung cancer, mammary cancer, cancer of the stomach, oral carcinoma, carcinoma of the pancreas, esophagus cancer, nasopharyngeal carcinoma, uterus carcinoma, prostate cancer, and carcinoma of gallbladder.

The 26th. each cancer vaccine of 23-25, it is used for the treatment of and has the people of HLA-A2402 as human leucocyte antigen.

The 27th. born of the same parents' poison T lymphocyte inductor, the major histocompatibility antigen complex body that comprises the 21st or 22 is as activeconstituents.

The 28th. the 27th born of the same parents poison T lymphocyte inductor, it is used for the treatment of and has the people of HLA-A2402 as human leucocyte antigen.

The 29th. the major histocompatibility antigen complex body tetramer, comprise major histocompatibility antigen and the 1st or 2 's peptide, perhaps be present in the tumour antigen epitope peptide on the 14th the antigen presenting cell.

The 30th. utilize one or more following (a)-(d) materials, by stimulating born of the same parents' poison T lymphocyte of peripheral blood lymphocyte acquisition:

(a) the 1st or 2 peptide;

(b) the 14th antigen presenting cell;

(c) the 21st or 22 major histocompatibility antigen complex body;

(d) the 29th the major histocompatibility antigen complex body tetramer.

The 31st. born of the same parents' poison T lymphocyte of the 30th, it obtains through the following steps: one or more (a)-(d) materials that utilize the 30th qualification, by stimulating peripheral blood lymphocyte, between major histocompatibility antigen complex body and/or its tetramer and born of the same parents' poison T lymphocyte, form complex body, and from complex body, separate born of the same parents' poison T lymphocyte.

The 32nd. the passive immunotherapy medicine, the born of the same parents' poison T lymphocyte that comprises the 30th or 31 is as activeconstituents.

The 33rd. the 32nd passive immunotherapy medicine, wherein cancer is an epithelial cancer.

The 34th. the 32nd or 33 passive immunotherapy medicine, wherein cancer is selected from large bowel cancer, lung cancer, mammary cancer, cancer of the stomach, oral carcinoma, carcinoma of the pancreas, esophagus cancer, nasopharyngeal carcinoma, uterus carcinoma, prostate cancer, and carcinoma of gallbladder.

The 35th. each passive immunotherapy medicine of 32-34, it is used for the treatment of and has the people of HLA-A2402 as human leucocyte antigen.

The 36th. the lymphocytic method of born of the same parents poison T that quantitatively HLA-A2402-limits in the peripheral blood, it comprises the following steps:

Make one or more following (a)-(d) materials act on peripheral blood:

(a) the 1st or 2 peptide;

(b) the 14th antigen presenting cell;

(c) the 21st or 22 major histocompatibility antigen complex body;

(d) the 29th the major histocompatibility antigen complex body tetramer, and

Born of the same parents' poison T lymphocyte in the quantitative peripheral blood or the cytokine that produces by such born of the same parents' poison lymphocyte.

The 37th. cancer therapy and/or improvement method comprise to having the people of HLA-A2402 as human leucocyte antigen and use one or more following (a)-(d) materials:

(a) the 1st or 2 peptide;

(b) the 14th antigen presenting cell;

(c) the 21st or 22 major histocompatibility antigen complex body;

(d) the 29th the major histocompatibility antigen complex body tetramer.

The 38th. cancer therapy and/or improvement method comprise the following steps:

From having HLA-A2402 as collecting monocytic cell part people's peripheral blood of patients of human leucocyte antigen,

Adopt one or more following (a)-(d) materials to cultivate the monocyte part:

(a) the 1st or 2 peptide;

(b) the 14th antigen presenting cell;

(c) the 21st or 22 major histocompatibility antigen complex body;

(d) the 29th the major histocompatibility antigen complex body tetramer, and

Will be wherein born of the same parents' poison T lymphocyte induced and/or the activated monocyte partly is back to blood samples of patients.

The 39th. induce the lymphocytic method of born of the same parents poison T, comprise to having the people of HLA-A2402 and use one or more following (a)-(d) materials as human leucocyte antigen:

(a) the 1st or 2 peptide;

(b) the 14th antigen presenting cell;

(c) the 21st or 22 major histocompatibility antigen complex body;

(d) the 29th the major histocompatibility antigen complex body tetramer.

The 40th. cancer therapy or improvement method comprise to having the people of HLA-A2402 as human leucocyte antigen and use born of the same parents' poison T lymphocyte of the 30th or 31.

The 41st. the 21st the major histocompatibility antigen complex body tetramer, wherein the tetramer is for comprising the HLA-A2402 molecule, B2M and the 1st or 2 's peptide perhaps is present in the complex body of the tumour antigen epitope peptide on the 14th the antigen presenting cell.

The 42nd. cancer vaccine, the major histocompatibility antigen complex body tetramer that comprises the 41st is as activeconstituents.

The 43rd. the 42nd cancer vaccine, wherein cancer is an epithelial cancer.

The 44th. the 42nd or 43 cancer vaccine, wherein cancer is selected from large bowel cancer, lung cancer, mammary cancer, cancer of the stomach, oral carcinoma, carcinoma of the pancreas, esophagus cancer, nasopharyngeal carcinoma, uterus carcinoma, prostate cancer, and carcinoma of gallbladder.

The 45th. each cancer vaccine of 42-44, it is used for the treatment of and has the people of HLA-A2402 as human leucocyte antigen.

The 46th. born of the same parents' poison T lymphocyte inductor comprises the 41st the major histocompatibility antigen complex body tetramer.

The 47th. the 46th born of the same parents poison T lymphocyte inductor, it is used for the treatment of and has the people of HLA-A2402 as human leucocyte antigen.

The invention effect

Epitope peptide provided by the invention is derived from the Ep-CAM molecule of wide expression on epithelial cancer cells, and can be restricted to born of the same parents' poison T lymphocyte identification of HLA-A2402, and HLA-A2402 is a modal human leucocyte antigen (HLA) type among the Japanese.As a result, this epitope peptide can be used as cancer vaccine and treats widely distributed HLA-A2402-positive human epithelial cancer.

The accompanying drawing summary

Fig. 1 illustrates the ELISOT analysis and evaluation result of polyclone peptide activated CTL clone.

Fig. 2 illustrates the tetramer coloration result of special polyclone of Ep-CAM peptide (A) and mono-clonal (B) CTL.

Fig. 3 illustrates Ep173-specificity-CTL-and clones feature.

Fig. 3 A illustrates ⁵¹Cr discharges analysis, shows that C27 (ctl clone that Ep173-is special) is at the cellular toxicity of T2-A24 in the presence of peptide Ep173 or control peptide EBV-LMP419.

Anti--HLA-A24 monoclonal antibody that Fig. 3 B illustrates ctl clone (C27) special to Ep173-is at the restraining effect of the cellular toxicity of HLA-A2402 positive cell line (PC9).

Fig. 3 C is illustrated in Ep173 and exists down, based on cold target inhibition analysis, and the cellular toxicity of C27.

The Ep-CAM that Fig. 4 illustrates PCR-based analyzes.

Fig. 5 illustrates the cellular toxicity of Ep173-specific CTL clone (C27) at multiple different cancerous cell line.

The best mode that carries out an invention

The present invention is described in detail as follows.

(I) epi-position/peptide

Structure

With reference to hla peptide in conjunction with the prediction (HLA Peptide Binding Predictions), BioInformatics ﹠amp; Molecular Analysis Section (BIMAS), ( Http:// bimas.dcrt.nih.gov/molbio/hla bind/index.html), this is that retrieval consists of 9-10 amino acid, has the reference point of HLA-A2402-in conjunction with the epitope peptide of primitive in aminoacid sequence, described sequence is derived from originating from the Ep-CAM albumen of wide expression on the epithelial cancer cells, as screening potential peptide is the result of epitope peptide, and peptide of the present invention is through being confirmed to be by the epitope peptide of born of the same parents' poison T lymphocytes (hereinafter referred to as " CTL ") identification.

More specifically, peptide of the present invention be following (1)-(4) any:

In the present invention, peptide is represented biological activity amino acid molecular chain, and wherein the alpha-amino group of adjacent amino acid residue is connected by peptide bond with carboxyl.

Except that above-mentioned peptide, peptide also comprises its salt and derivative, and this is because their biological activity does not have destroyed.The example of these derivatives comprises derivatives such as those glycosylations, amidation, phosphorylation, carboxylation, phosphonic acidsization, formylation, acidylate.Preferred salt is acid salt.The example of this salt comprises inorganic acid addition salts such as hydrochloric acid, phosphoric acid, sulfuric acid; And organic acid addition salt such as formic acid, acetate, propionic acid, tartrate.

(1)-(4) peptide that limits in is respectively derived from the Ep-CAM that originates from wide expression on the epithelial cancer cells, and the CTL identification that is limited by HLA-A2402-, or induces or further activate this CTL.Therefore, tumour antigen be induced or be activated to these peptides can be suitably as CTL-, i.e. cancer vaccine is treated or improved the human cancer with HLA-A2402.And these peptides can be used for preparing the special peptide of various tumour antigen epi-position.The application of peptide of the present invention is as described below.

(3) and the amino acid minimum of the mutant peptide that limits in (4) be about 5 usually, preferably be about 7.Amino acid whose maximum number is unrestricted, as long as the CTL identification that peptide can be limited by HLA-A2402-, and is about 12 usually, contain have an appointment 9 to about 11 amino acid whose mutant peptides be preferred.

By with reference to above-mentioned hla peptide in conjunction with the prediction (HLA Peptide Binding Predictions), design HLA-A2402-is in conjunction with the sequence of primitive, and the sequence of therefrom selecting those CTL that really limited by HLA-A2402-to discern or induce this CTL can obtain this mutant peptide.

HLA-A2402 is positive, and the Ep-CAM epitope peptide is for example identified by following method.

Lymphocyte separates the positive adult from HLA-A2402-, and with cell concn 2 * 10 ⁶/ ml is suspended in the RPMI1640 substratum that contains 10% human serum.Add wherein a kind of epi-position candidate's peptide to each cell suspending liquid with concentration 1 μ g/ml.With suspension at CO ₂37 ℃ of following incubations are 7 days in the incubator, and add IL-2 at the 7th day.Weekly 1 time repeat by candidate peptide stimulate and IL-2 stimulate circulation, to induce Ep-CAM-special CTL thereafter.

By ELISPOT analyze (Kuzushima K. etc., The Journal of ClinicalInvestigation, (1999), p.163-171) Vol.104 determines whether inductive epithelial cancer like this-special CTL is stimulated by epi-position candidate peptide.

Utilize above-mentioned candidate's peptide, the synthetic peptide library that contains 20 the amino acid whose peptides of having an appointment covers whole Ep-CAM albumen, the synthetic peptide is carried out ELISPOT to be analyzed, CTL shortens gradually to the peptide of its reaction, so that finally comprises about 9 to about 10 amino acid, thereby as epitope peptide of the present invention.

In addition, replace, have and be equal to active mutant peptide and obtain most probably about amino acid, if amino acid whose electric charge, the amino acid that solubility, hydrophilic/hydrophobic, character such as polarity are similar to those before replacing, the protein structure after then replacing is preserved.

Import disappearance, the method for adding (comprising insertion) and replacement is well-known, and example comprises WurmerShi technology (Science, 219,666 (1983)).

Peptide of the present invention can with carbohydrate, polyoxyethylene glycol, the complex body of additions such as lipid, the form of radio isotope derivative and polymkeric substance etc. is used.

(II) polynucleotide

Polynucleotide encoding the invention described above peptide of the present invention, and specifically each limits by following (5)-(8):

Except as otherwise noted, polynucleotide of the present invention comprise DNA and RNA.DNA comprises cDNA, genomic dna and synthetic DNA.RNA comprises mRNA, rRNA and synthetic RNA.Except that the polynucleotide with this base sequence, those are complementary in the polynucleotide of this polynucleotide and double-stranded polynucleotide are also included within.

The polynucleotide of base sequence shown in the SEQ ID NO:10 (one of polynucleotide of aminoacid sequence shown in the coding SEQ ID NO:1), with the polynucleotide of base sequence shown in the SEQ ID NO:11 (one of polynucleotide of aminoacid sequence shown in the coding SEQ ID NO:2) be partial sequence (Szala available from the Ep-CAM gene of people's tumor associated antigen GA733-2, S. etc., Proc.Natl.Acad.Sci.U.S.A. (1990) 87 (9), 3542-3546).

(7) and the sudden change polynucleotide that limit in (8) be that arbitrary those contain 15 or more, preferred 21 or more usually in the zone of code book invention epitope peptide, but common 45 or the polynucleotide of Oligonucleotide more.Wherein, it is preferred containing 27 polynucleotide to about 33 Nucleotide of having an appointment.

With the dna molecular is the representative example of polynucleotide molecule, " under stringent condition can with the dna molecular of dna molecule hybridize " can obtain by the described method of for example following document: " molecular cloning: laboratory manual " (volume such as Sambrook, Cold Spring HarborLaboratory Press, Cold Spring Harbor, New York, 1989) etc.In the present invention, " stringent condition " be meant, for example, at 6 * SSC, 42 ℃ of heating down in the solution of 0.5%SDS and 50% methane amide, and in the solution of 0.1 * SSC and 0.5%SDS 68 ℃ down during washing, observe the condition of positive hybridization signal.

Adopt following method, utilize known protein expression system, discerned by CTL or have the ability of inducing CTL having the peptide of expressing on the cell of HLA-A2402 by confirming, and select the sudden change polynucleotide of (7) and (8).

The sudden change polynucleotide have 3 ' poly A structure, in the polynucleotide few nucleotide of poly A without limits because do not influence the amino acid coding region of taking on tumour antigen.

When utilizing recombinant technology to prepare epitope peptide of the present invention, polynucleotide of the present invention can provide useful gene information.This polynucleotide can be used as nucleic acid reagent or standard polynucleotide.

(III) recombinant vectors

By importing polynucleotide of the present invention, obtain recombinant vectors of the present invention to suitable carriers DNA.

According to host cell kind and purposes purpose, can select carrier DNA suitably.Carrier DNA can be those naturally occurring carrier DNAs, perhaps those natural existence but part DNA is arranged from the carrier DNA to losing the unessential zone of propagation.The example of carrier DNA comprises that those originate from karyomit(e), the carrier DNA of episome and virus.More specifically, carrier DNA comprises those derived from bacterial plasmid, phage, transposon, yeast episome, insertion element, the yeast chromosomal element, virus is (for example, baculoviruses, corpora mammillaria polyoma vacuolating virus, SV40, vaccinia virus, adenovirus, fowlpox virus, Pseudorabies virus, retrovirus etc.) etc. carrier DNA, and combination, and those are derived from the carrier DNA of the gene element of plasmid and phage (for example, clay, phasmid etc.).

And expression vector and cloning vector can be used for biosynthesizing peptide of the present invention.

Recombinant vectors has the gene order of herbicide-tolerant polynucleotide and coding and duplicates gene order (for example, promotor, ribosome bind site with control information, terminator, signal sequence, enhanser etc.) element, and can make up these sequences by known method and prepare.

Polynucleotide of the present invention can insert carrier DNA by known method.For example, utilize suitable Restriction Enzyme, DNA can be connected at specific site with carrier DNA, and utilizes ligase enzyme to mix for reorganization.Perhaps, by connecting suitable joint, and insert polynucleotide to the multiple clone site of the carrier DNA that satisfies the purposes purpose and can obtain carrier DNA to polynucleotide of the present invention.

(IV) transformant

Utilize known method, the above-mentioned recombinant vectors that contains polynucleotide by importing is to known host cell, such as, (for example, K12), the bacterium that belongs to Bacillaceae (for example for intestinal bacteria (Escherichia coli), MI114), yeast (for example, AH22), insect cell (for example, the Sf cell), zooblast is (for example, the COS-7 cell, the Vero cell, Chinese hamster ovary celI etc.) etc., can obtain transformant of the present invention.

In view of good gene stability, gene integration to chromosomal method is preferred gene transfer method.Utilizing the automatic dubbing system of extranuclear gene is simple available method.Carrier DNA is transferred to host cell can be undertaken by the standard method that is disclosed in for example following document: " molecular cloning: laboratory manual " (Sambrook etc., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989) etc.Example comprises calcium phosphate transfection more specifically, DEAE-dextran mediation transfection, and microinjection, the transfection of cation lipid mediation, electroporation, transduction is scraped and is carried (scrape loading), trajectory importing and infection etc.

(V) preparation method of epitope peptide

Peptide of the present invention can pass through known method of peptide synthesis, is synthesized such as solid phase method of peptide synthesis etc.This method of peptide synthesis comprises that those are disclosed in the method in " peptide synthesizes (Peptide Synthesis) " (Maruzen, 1975) and " peptide synthesizes (Peptide Synthesis) " (Interscience, New York, 1996).Perhaps, epitope peptide of the present invention can utilize known chemosynthesis equipment, and the peptide synthesizer of producing such as Applied Biosystems prepares.

In addition, the preparation method of peptide of the present invention comprises the following steps: to cultivate the transformant of the invention described above, and collects peptide of the present invention from this culture.

Utilization by inferior cultivation or batch culture, is carried out this cultivation to the substratum that host cell is fit to.With required amount production, this is based on the peptide amount of producing inside and outside the transformant, and/or the CTL inducibility up to peptide of the present invention in lasting cultivation, and described ability is by one of effect of the peptide demonstration of the present invention of transformant production.

After from substratum, collecting, preferably be further purified peptide of the present invention.Purifying can be undertaken by known method, such as sieve chromatography, and ion exchange chromatography, chromatographic techniques such as affinity chromatography; Utilize ammonium sulfate and/or alcohol etc., based on the combination of the hierarchical approaches of solvability difference.The carrying out of this purifying can be induced based on identification of the peptide by CTL or the CTL by peptide, more specifically for example, and by the IFN-γ output of CTL.

Perhaps, the peptide of the present invention in the substratum utilizes polyclone or monoclonal antibody to be reclaimed by specific adsorption, and the production of described antibody is based on the aminoacid sequence of peptide of the present invention, and is specific to this aminoacid sequence.

(VI) antigen presenting cell

Antigen presenting cell of the present invention is by with peptide pulse dendritic cell of the present invention, scavenger cell, antigen presenting cell such as bone-marrow-derived lymphocyte and obtaining.Antigen presenting cell of the present invention can be expressed in its surface and peptide bonded HLA of the present invention, and has the ability that stimulates CTL.

" pulse " is unrestricted, and can be by for example in the substratum of the peptide of the present invention that contains the 1-10 μ g/ml that has an appointment, about 30 ℃ of this antigen presenting cells of following incubation of about 20-about 30 minutes to about 1 hour and carrying out.The tumor antigen peptide that the CTL that is limited by HLA-A2402-discerns is presented thus on the antigen presenting cell surface.

Tumour antigen is meant to be present in the tumour cell and is discerned by tumour-special CTL, or further induces CTL or/and activate albumen, polypeptide or the peptide of CTL.And tumor antigen peptide is meant the peptide that produces by the tumour antigen that decomposes in the tumour cell, and as the result who is attached to the HLA molecule, when presenting on cell surface, this peptide is discerned by CTL, induces CTL, or/and activate CTL.

Epitope sequences is derived from the aminoacid sequence in the tumour antigen, and discerned by TS CTL, or further induces or activate CTL, is called tumour antigen epi-position (tumour antigen determinant).

In this manual, " identification " expression understanding and differentiation target and other materials, and for example, be attached to the target of being familiar with.In this manual, " CTL tumor cell or tumour antigen epitope peptide " expression is by t lymphocyte receptor, and CTL is attached to human leucocyte antigen (HLA) (HLA) or tumor antigen peptide.

" activation " expression further strengthens or stimulates material or the state with certain activity or effect.Particularly, " CTL is activated " expression CTL produces effector, such as, INF-γ, or demonstration when CTL discerns the epitope peptide of being presented by HLA, is discerned these target cells at the cellular toxicity of target cell.

" induce " expression not cause this activity or effect from having certain material or state active or effect substantially.Particularly, the outer or body internal specific of " CTL that inducing antigen is special " expression differentiation and/or vegetation is discerned certain antigenic CTL.

Antigen presenting cell with its pulse epitope peptide of the present invention can be used as cancer vaccine.Antigen presenting cell can be further used for producing the CTL that is specific to epidermal derived cancer, this CTL in the quantitative human peripheral etc.

(VII) major histocompatibility antigen complex body

Major histocompatibility antigen complex body of the present invention be major histocompatibility antigen and peptide of the present invention or present pulse with the antigen presenting cell of peptide of the present invention complex body between the lip-deep tumour antigen epitope peptide.

Among the present invention, major histocompatibility antigen is that the peptide of being discerned by the T lymphocyte is presented molecule to t lymphocyte antigen receptor, and is the human leucocyte antigen (HLA) of being discerned by CTL with epitope peptide (HLA).

More specifically, major histocompatibility antigen complex body of the present invention (hereinafter referred " MHC ") is the complex body between MHC I type human leucocyte antigen (HLA) (HLA) and the epitope peptide that maybe can be induced CTL by CTL identification, and described HLA is expressed in the target cell surface of being discerned by CTL with epitope peptide.This species complex comprises that those are by β2Wei Qiudanbai bonded complex body.

The special CTL epitope peptide of Ep-CAM-is the peptide of specific site in Ep-CAM albumen, and the antigenic determinant that is discerned by CTL, and immunity is attached to the antigen receptor of CTL.Further, the CTL epitope peptide can be eliminated cancer cells by the cell of direct attack expression Ep-CAM molecule.

Especially, MHC of the present invention is meant the complex body between the defined epitope peptide of being discerned by CTL in the HLA-A2402 molecule that is expressed on the target cell and the Ep-CAM albumen, and can be meant this species complex that forms with β2Wei Qiudanbai.

HLA-A2402 among the present invention is meant the antigenic hypotype of I type in the human leucocyte antigen (HLA), the A-24 polymorphism, and HLA-2402 divides the subrepresentation antigen presenting cell lip-deep HLA-A2402 gene expression product.

Available HLA-A2402 molecule is that purifying is expressed arbitrary HLA-A2402 molecule of using the E.coli culture from HLA-A2402, by the HLA-A2402 molecule of metastatic gene to the target cell forced expression, and the natural HLA-A2402 molecule that is present on the target cell.And HLA-A2402 molecule of the present invention comprises the fragment and the heavy chain of HLA-A2402 molecule.

MHC of the present invention depends on that its element can be by multiple diverse ways preparation, and by for example peptide of the present invention, HLA-A2402 molecule and β2Wei Qiudanbai being suspended in such as damping fluids such as Tris, and form about 72 hours of the about 10 ℃ of about 24-of this suspension of following incubation of about 4-.

MHC can the tetramer.The MHC tetramer is the poly complex body of four inferior complex bodys, and each inferior complex body comprises Ep-CAM epitope peptide and the HLA-A2402 molecule of being discerned by CTL (β2Wei Qiudanbai bonded HLA-A2402 molecule usually).

The MHC tetramer is by biotinylation MHC, and with 1: 4 obtain than combination chain avidin and biotinylated MHC.Perhaps, the MHC tetramer makes vitamin H be bonded to such site by adding the C end of vitamin H binding site to the HLA molecule after forming MHC, then with 1: 4 obtain than combination chain avidin and biotinylated MHC.

More specifically, the MHC tetramer can for example following mode prepare.Be preparation MHC, the E.coli expression of recombinant proteins system mass production that HLA-A2402 molecule heavy chain and β2Wei Qiudanbai utilize MHC to express, and purifying.Add the aminoacid sequence of identification vitamin H ligase enzyme in advance at HLA-A2402 heavy chain C end.The HLA-A2402 heavy chain and the β2Wei Qiudanbai of purifying are dissolved in 8M urea respectively.(pH 8.0 to 200ml refolding damping fluid, 100mMTris-HCl, 400mM L-arginine-HCl, 2mM EDTA, 0.5mM oxidized form Triptide, 5mM reduced glutathione), utilize the syringe of No. 27 pins respectively, adding 12mg has peptide of the present invention, 18.6mgHLA-A2402 heavy chain and the 13.2mg β2Wei Qiudanbai of aminoacid sequence shown in the SEQ ID NO:1 (RYQLDPKFI).Mixture stirred 48-72 hour in 10 ℃ bath in temperature maintenance, formed to promote MHC.The refolding damping fluid that contains MHC subsequently in temperature maintenance in 4 ℃ bath to 1.8L distill water dialysis 24 hours, and the refolding damping fluid of dialysis utilizes Centriprep 10 (Millipore Corporation, Bedford MA) to be concentrated into 2ml.By gel permeation chromatography, (Amersham Pharmacia Biotech AB UppsalaSweden) separates, thereby obtains the required MHC tetramer to utilize Superdex 200HR at the about 45kD of the molecular weight eluted MHC in place.

For preparing biotinylated MHC subsequently, utilize vitamin H ligase enzyme (AVIDITY, LLC, Denver CO), vitamin H is bonded to the specific site of HLA-A2402 heavy chain C end.Vitamin H bonded MHC utilizes Superdex 200HR and purifying by gel permeation chromatography.

Then; the streptavidin of PE-mark (Molecular Probe; Eugene OR) and the biotinylated MHC of purifying with 1: 4 mixed in molar ratio; and utilize Superdex 200HR to separate by gel permeation chromatography at the MHC tetramer of aminoacid sequence shown in the eluted SEQ of the containing ID NO:1 of the about 480kD of molecular weight place (RYQLDPKFI); be concentrated into about 3mg/ml by Centricon10 (Millipore Corporation), and maintain 4 ℃.Such as sodium azide, EDTA, sanitass such as leupeptin and Pepstatin can add wherein.

In above-mentioned each preparation process, it is desirable to by such as standby albumen of known method purifying and peptides such as gel permeation chromatographies.

MHC of the present invention can be used as cancer vaccine.And it can be used for preparing the special CTL of epithelial cancer, and this CTL that is used for quantitative human peripheral.

(VIII) cancer vaccine, CTL inductor and gene therapy medicament

Cancer vaccine and CTL inductor

Peptide of the present invention can be advantageously used for the activeconstituents of cancer vaccine, with the vaccine passive immunotherapy.This cancer vaccine can be used for having the philtrum of HLA-A2402.

That is, give to have the CTL that HLA-A2402 uses inducing peptide of the present invention as the cancer patients of human leucocyte antigen or activates the HLA-A2402-restriction of specific recognition Ep-CAM, make treatment or improve disease such as epithelial cancer and become possibility thus.

Because cancer patients's CTL is one group of cell of discerning two or more tumour antigens, therefore utilize multiple epitope peptide as cancer vaccine sometimes Billy have better effect with single kind epitope peptide.Peptide of the present invention can multiple alone or in combination (two or more) utilize.

The mRNA that contains the Ep-CAM of peptide of the present invention is expressed in lung cancer cell line (QG56, LU99, LC99A, LC1-sq, LC65A and PC-9), colorectal carcinoma cell line, stomach cancer cell system (MKN28, MKN45), cancer cell of oral cavity system (HSC-2), breast cancer cell line, leukemia cell system (K562) etc.The expression of Ep-CAM is also from lung cancer, large bowel cancer, and cancer of the stomach is observed in mammary cancer or oral cancer patient's the tissue.Therefore, peptide of the present invention is useful to treating or improving this cancer.

No matter peptide of the present invention still makes up with multiple different carriers separately, can be made into preparation.Preparation can adopt oral or parenteral form.Usually, parenteral form is preferred.The example of parenteral administration comprises the subcutaneous injection agent, intramuscularly agent, intravenous injection agent, suppository etc.

Utilize and can not suppress the active pharmaceutically acceptable vehicle of epitope peptide of the present invention, can prepare oral preparations.The example of this vehicle comprises starch, N.F,USP MANNITOL, lactose, Magnesium Stearate, Mierocrystalline cellulose, polymeric amino acid, albumin etc.

Utilize and can not suppress the active pharmaceutically acceptable carrier of epitope peptide of the present invention, can prepare parenteral administration.The example of this carrier comprises water, sodium-chlor, D-glucose, ethanol, glycerine, DMSO etc.As needs, parenteral administration can further contain albumin, wetting agent, emulsifying agent etc.For the immunity of irritation cell mediation, epitope peptide preferably is used in combination with suitable adjuvant.Peptide of the present invention can natural or salt form use.The example of pharmacologically acceptable salt comprises all example hydrochloric acids, the salt of mineral acids such as phosphoric acid, and such as acetate, organic acid salt such as tartrate.

By preparing peptide with the antibody of compound, immune identification polypeptide etc., the activity of adjustable abridged edition invention peptide, described compound self or interact with HLA-A2402 and to strengthen peptide by the identification of CTL.

Pulse also can be advantageously used for cancer vaccine with the antigen presenting cell and the major histocompatibility antigen complex body of peptide of the present invention.The dosage form of this vaccine, the target patient, the target cancer, effects etc. are the same with the cancer vaccine that contains peptide of the present invention.

Methods of treatment

Peptide of the present invention, antigen presenting cell and major histocompatibility antigen complex body have the man-hour of HLA-A2402 as human leucocyte antigen when delivering medicine to, and induce or activate the CTL of the special HLA-A2402-of Ep-CAM-restriction, thus treatment or improve epithelial cancer.

The dosage of peptide of the present invention is changed by CTL identification degree with peptide, and based on active epitope peptide, per day for adults can be for example, the about 100mg of about 0.01mg-, and the about 30mg of preferably about 0.1mg-.Depend on the administration purpose, spacing of doses can be individual patients and is selected suitably.

Perhaps, import the monocyte part to the blood samples of patients circulation, can realize effective cancer immunity inoculation, described part has been separated the peripheral blood from the patient, and cultivates to induce and/or to activate CTL with peptide of the present invention.To culture condition,, selected feasible adult administration 10 such as the concentration of monocyte and peptide of the present invention ⁸-10 ¹⁰CTL/ days.This condition can be determined by simple test is convenient.Material with lymphocytic hyperplasia ability such as interleukin-2, can add in culturing process.

The dosage of antigen presenting cell to the adult for for example, about 10 ⁶-10 ⁹Cell/sky, preferred 10 ⁷-10 ⁸Cell/sky.And, separate from the monocyte part of patient's peripheral blood and the coculture of antigen presenting cell of the present invention by importing to the blood samples of patients circulation, can realize effective cancer immunity inoculation.

The major histocompatibility antigen complex body is for example 1-100mg/ days to the adult, preferred 5-50mg/ days.And, can realize effective cancer immunity inoculation by importing separation to the blood samples of patients circulation from the monocyte part of patient's peripheral blood and the coculture of major histocompatibility antigen complex body of the present invention.

The gene therapy medicament of cancer

Polynucleotide of the present invention (comprising complementary strand), as for example lung cancer, large bowel cancer, cancer of the stomach, mammary cancer, the gene therapy medicament of oral carcinoma etc. is useful.

For cancer therapy, the carrier that contains polynucleotide of the present invention can directly import in the body, or imports the cell of collecting from the people, and described cell is back in the body again.Carrier is unrestricted, and can be selected from those and become known for Vectors in Gene Therapy, such as retrovirus, and adenovirus, vaccinia virus etc., wherein retrovirus is preferred.This virus is replication defective.

But polynucleotide of the present invention are by microinjection technology transfered cell, and polynucleotide are encapsulated in the liposome and shift in described technology.

Therefore, gene therapy for cancer medicine of the present invention can be any form: independent polynucleotide of the present invention, contain the recombinant vectors of polynucleotide of the present invention, and make the liposome of polynucleotide encapsulation of the present invention etc.

The dosage of polynucleotide of the present invention is changed by the degree that CTL discerns with the peptide by polynucleotide encoding.For example, adult's suitable dose, based on the amount of the part polynucleotide of code book invention epitope peptide, about 0.1 μ g-about 100mg/ days, preferred about 1 μ g-about 50mg/ days.The administration of this dosage can every several days once to per several months once.

Polynucleotide of the present invention can with cytokine, such as IL-2, or use, thereby strengthen the expression of polynucleotide with the interactional combinations of substances of polynucleotide of the present invention.

(IX) born of the same parents' poison T lymphocyte

Utilize at least a material in following (a)-(d),, can obtain born of the same parents' poison T lymphocyte of the present invention by stimulating peripheral blood lymphocyte:

(a) peptide of the present invention,

(b) antigen presenting cell of the present invention;

(c) major histocompatibility antigen complex body of the present invention and

(d) the major histocompatibility antigen complex body tetramer of the present invention.

The induction method of CTL of the present invention is that at least a material of peripheral blood lymphocyte in (a)-(d) cultivated about 7-about 14 days under about 37 ℃ in preferably containing the RPMI1640 substratum of human serum.When peripheral blood lymphocyte is stimulated by major histocompatibility antigen complex body (hereinafter referred to as " MHC ") or its tetramer, CTL induced and with MHC or tetramer combination.Therefore, by suitable method, CTL separates from the MHC or the tetramer.

Particularly, CTL can obtain by for example following preparation method.

When stimulating with epitope peptide or antigen presenting cell

The lymphocyte that separates from peripheral blood is cultivated about 7-about 10 day under about 37 ℃ with the antigen presenting cell of this peptide with peptide of the present invention or pulse in CO2gas incubator.Then, preferably adding IL-2, PHA, behind the anti-CD 3 antibodies etc., about 14 days of the about 7-of incubation, thus stimulate and propagation CTL.

Stimulate with described peptide or antigen presenting cell, optional following with stimulations such as IL-2 implemented about 3 times, thereby obtains requisite number purpose CTL.

Like this form of suspension that the special CTL of the epithelial cancer that obtains for example can be in the PBS that contains human serum etc. is as the passive immunotherapy drug use of epithelial cancer etc.The form of passive immunotherapy medicine is generally injection, such as the intramuscularly agent, and subcutaneous injection agent, intravenously infusion etc.

When stimulating with MHC or its tetramer

Dyeing separates

Lymphocyte is separated from peripheral blood etc., and reacted about 30-about 60 minutes down for about 25 ℃ with the MHC of suitable concn or the MHC tetramer about 4-in PBS for example.Labeling dye such as FITC or PE, adds in the reaction mixture, so that be attached to MHC or the tetrameric CTL dyeing of MHC.Subsequently, painted CTL utilizes flow cytometer or microscope to separate.

Fixedly separated by MHC

Utilize its surface to be fixed with MHC or the tetrameric sterile plate of MHC, reacted separate in the lymphocyte of peripheral blood and plate the fixed MHC or the MHC tetramer to about 30-about 60 minutes about 25 ℃ times at about 4-.Wash other not behind combination and the buoyant cell, the special CTL of the epithelial cancer of leaving on the plate is suspended in the new substratum.The special CTL of isolating epithelial cancer like this can the T cell stimulatory agents, such as anti-CD 3 antibodies, and PHA, stimulations such as IL-2, thus CTL is bred to the required number of passive immunotherapy medicinal application.

The use of magnetic bead

Zhi Bei biotinylated MHC is bonded to the magnetic bead of streptavidin mark as mentioned above, with preparation binding substances (hereinafter referred to as " MHC-magnetic bead ").Subsequently, lymphocyte separates from peripheral blood etc., and adds the MHC-magnetic bead with suitable concentration, makes lymphocyte: the ratio of pearl becomes 1: 5-20, then reaction.

When the testing tube that contains the special CTL of the epithelial cancer that is bonded to the MHC-magnetic bead places magnetic field, be adsorbed to magnetic force one side of testing tube inwall with pearl bonded CTL.

Owing to be attached to the testing tube inwall with described pearl bonded CTL, other cells are rinsed.Thereafter, testing tube shifts out from magnetic field, and the CTL of remaining antigen-specific is suspended in the new substratum in the testing tube.

The special CTL of isolating epithelial cancer like this can the agent of T LS, such as anti-CD 3 antibodies, and PHA, stimulations such as IL-2, thus CTL is bred to the required number of passive immunotherapy medicinal application.

Treatment or improve method for cancer

CTL of the present invention, when its during as the supreme patient with canceroderm of passive immunotherapy drug administration, can treat or improve cancer.

Dosage depends on that the administration purpose changes with the patient.For example, adult's suitable dose about 10 ⁷-Yue 10 ¹¹Cell/sky, preferred about 10 ⁸-Yue 10 ¹⁰Cell/sky.The administration of this dosage can every several days once to per several months once.

(X) method of quantitative CTL

In the cancer therapy management, comprise suitable use anticarcinogen and chemotherapeutic, importantly know, whether the special CTL of epithelial cancer is present in the excessive risk cancer patients (patient that the immunity that is caused by cancer cells or cancerous tissue reduces, complication patient, gerontal patient, infant patients, pregnant woman patient) peripheral blood in.The CTL of the HLA-A2402-restriction that epithelial cancer is special can be quantitative by following method.

Method comprises makes that arbitrary material acts on peripheral blood sample in following (a)-(d), and the quantitatively CTL in the sample or the step of consequent cytokine:

(a) peptide of the present invention,

(b) antigen presenting cell of the present invention,

(c) MHC of the present invention and

(d) the MHC tetramer of the present invention.

Compare with income value and by the quantitative value of same procedure (except that the solution of the CTL that uses the HLA-A2402-restriction that contains concentration known), thereby calculate the CTL concentration of HLA-A2402-restriction in the peripheral blood sample.

By the CTL of one or more (a)-(d) material inductive HLA-A2402-restriction, or the CTL cytokine of producing thus, but for example quantitative assay is as follows.

When using epitope peptide

The lymphocyte that separates from peripheral blood is stimulated with peptide of the present invention, and measure the number of inductive CTL or thus the CTL generation such as interferon-(IFN-γ), the amount of cytokines such as interleukin (comprising chemokine).Concrete grammar is described below, and is example with the situation of IFN-γ.

IFN-γ produces the quantitative of cell in the born of the same parents

To separate lymphocyte from peripheral blood with cell concn 2 * 10 ⁶/ ml is suspended in the RPMI1640 substratum that contains 10% human serum, and adds CTL epitope peptide of the present invention with concentration 1mg/ml.In addition, add the intracellular protein transport inhibitors,, then in CO2gas incubator, cultivated 5-6 hour under 37 ℃ such as brefeldin A.After the cultivation, cell fixation is carried out membrane permeation and is handled, and reacts with fluorescently-labeled resisting-IFN-gamma antibodies.Utilize flow cytometer etc. that the IFN-γ positive cell in the CD8 positive lymphocyte is carried out quantitative assay.

The CD8 positive cell quantitatively

The CD8 positive cell of mark can be quantitative according to the method (except that utilizing anti--CD8 antibody to replace the anti-IFN-gamma antibodies of fluorescence-mark) of IFN-γ production cell in the above-mentioned quantitative born of the same parents.

Quantitative (the ELISPOT analysis) of cytokine

Spent the night with anti--IFN-γ monoclonal antibody bag under 4 ℃ in 96 holes MultiScreen-HA plate (Millipore).After the PBS washing hole, the lymphocyte that separates from peripheral blood is seeded in the hole.Epitope peptide places in the hole, and at 5%CO ₂Cultivated 20 hours down for 37 ℃ in the incubator.Next day, plate washs with the PBS that contains 0.05%Tween-20, and at room temperature reacts 90 minutes with anti--IFN-γ rabbit anteserum, and the anti-rabbit igg lowlenthal serum with the peroxidase mark reacted 90 minutes then.In addition, the 0.1M sodium acetate buffer (pH 5.0) that will contain 3-amino-9-ethyl carbazole (Sigma) and 0.015% superoxol places the hole, and at room temperature reacts 40 minutes.IFN-γ point is visual and count with stereoscopic microscope.

Quantitatively secrete the method for the cytokine in culture supernatants

To separate lymphocyte from peripheral blood with cell concn 2 * 10 ⁶Individual cell/ml is suspended in the RPMI1640 substratum that contains 10% human serum, and adds CTL epitope peptide of the present invention with concentration 1 μ g/ml.In CO2gas incubator, cultivated 24-48 hour down for 37 ℃.After the cultivation, collect supernatant liquor, and utilize commercially available ELISA test kit (for example, the people IFN-γ ELISA of ENDOGEN) to measure the concentration of IFN-γ in the supernatant liquor.

When using the MHC tetramer

Lymphocyte separates from peripheral blood etc., and with concentration be that the MHC tetramer (fluorescent mark has streptavidin) of 1-100 μ g/ml is 37 ℃ of reactions 15 minutes down.Dye by adding antibody with MHC bonded CTL, described antibodies to MHC bonded CTL, and be marked with another fluorescence dye.Utilize flow cytometer or microscope, to painted CTL counting.

Embodiment

Hereinafter, with reference to embodiment, with more detailed description the present invention, but the present invention is not limited to

Embodiment.

Embodiment 1

1) contributor and clone

All contributors know research project and the purpose thereof by the approval of ethics committee of Aichi Cancer center fully.The positive healthy contributor of the HLA-A2402 of peripheral blood sample after of test usefulness available from 5 informed consents.From peripheral blood sample,, isolate peripheral blood lymphocytes (PBMC) by Ficoll density gradient centrifugation available from each contributor.

Large cell carcinoma clone LU99 (JCRB0080) is as the human lung cancer cell line; People's epidermis shape cancerous cell line HSC-2 (JCRB0622); Epidermis shape cancerous cell line MKN28 (JCRB0253) and MKN45 (JCRB0254) are as SGC-7901; And epidermis shape cancerous cell line COLO320DM (JCRB0225 or ATCC:CCL-220) is available from JCRB cell bank (Ministry of Health, Labour and Welfare:http//Cellbank.nihs.go.jp) as people's rectum cancer cell.Epidermis shape cancerous cell line LC-1/sq (RCB0455) as the human lung cancer cell line available from the Riken cell bank.

The LC/sq cell maintains in 45%RPMI1640 substratum and the 45%HamShi F12 substratum (Sigma product), and described substratum prepares by adding the liquid nutritional fill-in, and described nutritional supplement comprises 10%FCS, L-glutaminate, penicillin, Streptomycin sulphate, and kantlex.COLO320DM cell and MKN28 maintain in the DMEM substratum (Sigma product).The K562 cell maintains in the IMDM substratum (Sigma product), and described substratum prepares by adding the liquid nutritional supplement, and described fill-in comprises 10%FCS (foetal calf serum, the LifeTechnology product), L-glutaminate, penicillin, Streptomycin sulphate and kantlex.

Other cancerous cell lines are cultivated in the RPMI1640 substratum, and described substratum prepares by adding the liquid nutritional supplement, and described fill-in comprises 10%FCS, and 2 * 10 ^-3The M L-glutaminate, 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates, 100 μ g/ml kantlex and 5 * 10 ^-5M beta-mercaptoethanol (as perfect medium).

The HLA-A2402 gene imports 174CEM.T2 (hereinafter being called " T2 cell "), provides the cell (hereinafter being called " T2-24 cell ") of presenting the HLA-A2402 binding peptide and is delivery cell as peptide.The T2-24 cell cultures is containing 10% foetal calf serum, L-glutaminate, penicillin, the IMDM substratum of Streptomycin sulphate and G418 (Gibco product) (the DulbeccoShi substratum that IscoveShi revises: the Gibco product).

According to " tissue antigen " (Tissue Antigens, 59:502-511, (2002)) method described in, negative QG56 clone of HLA-A2402-and negative A549 (JCRB0076) clone of HLA-A2402-are with the retroviral infection of coding HLA-A2402.In order to detect the cell of infection, the QG56 cell that infects maintained contain in the perfect medium of tetracycline that final concentration is 0.6 μ g/ml, and called after QG56-A24.Equally, the A549 cell of infection maintains and contains in the perfect medium of tetracycline that final concentration is 0.9 μ g/ml, and called after A549-A24.

2) peptide is synthetic

(accession number: potential HLA-A2402-bonded peptide is by computer forecast, based on from World Wide Web Sheng Wuxinxixue ﹠amp M33011) at Ep-CAM; Analysis of molecules part (BIMAS:BioInformatics and Molecular AnalysisSection) (htt: //bimas.dcrt.gov/molbio/hla_bind) the half time of obtainable hla peptide complex body expectation dissociates, plan (HLA Peptide BindingPrediction Program) according to hla peptide in conjunction with prediction, and identified.

Synthetic based on predict the outcome 7 kinds of peptides selecting of Ep-CAM epitope peptide by PepSet (Mimotope product).If desired, the gained peptide is dissolved in the 100 μ l dimethyl sulfoxide (DMSO), and further with 40% dilution in acetonitrile to 0.1M (pH 7.4).

It is 1 μ mol that the turnout of each peptide is estimated.

7 kinds of synthetic peptide difference called after Ep31, Ep173, Ep185, Ep250, Ep225, Ep296, and Ep304.Table 1 illustrates the synthetic peptide sequence of Ep-CAM.

Table 1

The peptide name	Aminoacid sequence	Amino acid position	Amino acid length	Score ^a	%MFI increases ^b
The peptide name	Aminoacid sequence	Amino acid position	Amino acid length	Score ^a	%MFI increases ^b	Ep31	NYKLAVNCF	31-39	9	120	85
Ep173	RYQLDPKFI	173-181	9	150	102	Ep31	NYKLAVNCF	31-39	9	120	85
Ep173	RYQLDPKFI	173-181	9	150	102	Ep185	LYENNVITI	185-193	9	75	79
Ep225	LFHSKKMDL	225-233	9	20	29	Ep185	LYENNVITI	185-193	9	75	79
Ep225	LFHSKKMDL	225-233	9	20	29	Ep250	YYVDEKAPEF	250-259	10	198	57
Ep296	KYEKAEIKEM	296-305	10	83	24	Ep250	YYVDEKAPEF	250-259	10	198	57
Ep296	KYEKAEIKEM	296-305	10	83	24	Ep304	EMGEMHREL	304-312	9	5	16

Remarks:

^a: from expectation half time of HLA-A24 molecular dissociation (minute), by visit World Wide Web Sheng Wuxinxixue ﹠amp; Analysis of molecules part (BIMAS) hla peptide obtains in conjunction with prediction.

^b: based on the MHC stability analysis, the synthetic peptide of assessment is active to the combination of HLA-A24 molecule.%MFI represents average fluorescent strength.

In addition, human immunodeficiency virus-1 (HIV-1) env peptide RYLRDQQLL (called after ENV584, J.Immunol, 159:6242-6252, residue 584-592) and EBV latent membrane protein 2 peptides (the potential film of EBV) (called after EBV-LMP419 1997:, J.Immunol, 158:3325-3334,1997: residue 419-427) synthetic with comparing (Toray Industriesresearch center company).

3) cell dyeing and flow cytometry analysis

Utilization is expressed in the anti-mouse IgG (ab ') of anti-HLA-A2402 monoclonal antibody (One Lambda company product) on the cell surface and FITC mark ₂Fragment (Immunotech product) is tested by the indirect immunofluorescence antibody analysis.According to step described in " Blood, 98:1872-1881,2001, Science, 274:94-96,1996 ", preparation MHC/ peptide tetramer.

CD8 positive T cell system or its are cloned the HLA-A2402 tetramer (being called the HLA-A2402/Ep173 tetramer) with phycoerythrin (the PE)-mark that contains Ep-CAM peptide and Ep173, or contain the HLA-A2402 tetramer (the being called the HLA-A2402/ENV584 tetramer) dyeing of the PE mark of HIV-1 peptide and ENV584.

Utilize FACSCalibur (Becton, Dickinson and Company product) that painted cell is carried out flow cytometry analysis, and by CellQuest software (Becton, Dickinson andCompany product) analytical data.

The MHC stability analysis

For assessing the HLA-A2402 joint efficiency of synthetic peptide, step (Blood according to Kuzushima etc., 98:1872-1881,2001), utilize T2-A24 cell (it is transfected to T2[174 * CEM.T2:ATCC accession number: CRL-1992 for the plasmid of expressing the HLA-A2402 molecule] clone) to carry out the MHC stability analysis.

Particularly, be that each peptide of 10 μ M contains T2-A24 cell (2 * 10 at 200 μ l with concentration ⁵Individual cell), 0.1%FCS and 5 * 10 ^-526 ℃ of following incubations are 16 hours in the RPMI1640 substratum of M beta-mercaptoethanol, then continue incubations 3 hours down at 37 ℃.Behind the incubation, cell surface HLA-A2402 molecule is with above-mentioned 3) in anti-HLA-A2402 monoclonal antibody and the antibody staining of FITC mark.Determine expression level by FACSCalibur, and record average fluorescent strength (MFI).

%MFI increases by following formula calculating:

%MFI increases=100 * not (with the MFI-of the group of peptide administration not with the MFI of the group of peptide the administration)/MFI of the group of peptide administration (not with)

Table 1 illustrates the result that relevant %MFI level increases.As shown in table 1, the HLA-A2402 expression level on the cell surface increases most of peptides, shows that these peptides are bonded to cell surface, makes MHC stable thus.

Particularly, in above-mentioned peptide, Ep173 (RYQLDPKFI) shows the highest avidity to HLA-A2402.Ep304 (EMGEMHREL) is owing to show that minimum avidity does not continue other tests.

5) special CTL of Ep-CAM peptide and clone's production thereof

Step (J.Immunol, 170:4069-4076) production peripheral blood lymphocytes deutero-dendritic cell (DC) according to Dauer etc.

More specifically, the cell that is attached to plastics is separated from the positive healthy volunteer's of HLA-A2402 peripheral blood lymphocytes, and is incubated at and contains 5% hot deactivation human blood serum, 10 μ g/ml recombination human interleukin-4 (hIL-4, R﹠amp; D systems Company product) and 50ng/ml macrophage colony stimulating factor of recombinant human granulocyte (hGM-CSF, R﹠amp; D systemsCompany product) RPMI1640 substratum.After the incubation 1 day, for the cell growth, PeproTech Inc product) and 1 μ M prostaglandin E2 (CaymanChemical Company product) add 50ng/ml IL-1 β (PeproTech, Inc product), 50ng/ml recombination human tumor necrosis factor-alpha (hTNF-α:.After 2 or 3 days, collecting cell is used for the dendritic cell of the monocyte derived of antigen-presenting.

The cell of producing is obviously expressed the cell-mediated antigen of dendron, such as CD1a, and CD80, CD83, CD86 and HLA II type molecule.

In addition, utilize CD8 microballon (Milteny Biotec product), from identical donor, isolate the CD8 positive t lymphocytes.

Subsequently, pulse was synthesized peptide 2-4 hour with each Ep-CAM under the self-derived dendritic cell room temperature, and this peptide is containing 5 * 10 ^-5Concentration is 10 μ M in the AIM-V substratum of M beta-mercaptoethanol (Gibco product), then radiation (33Gy).

Then, dendritic cell (1 * 10 ⁵Cell) with CD8 positive t lymphocytes (1 * 10 ⁶Lymphocyte) cultivation is containing 10% human blood serum, 25ng/ml recombinant human IL-7 (R﹠amp altogether; D systemscomapany product) and 5ng/ml recombinant human IL-12 (R﹠amp; In the culture tube of RPMI1640 substratum D systems company product).

After cultivating 7 days, add 1 * 10 ⁵The self-derived dendritic cell of the peptide pulse of individual above-mentioned preparation come irritation cell.After continue cultivating 7 days, with same steps as irritation cell 3 times.After stimulating again at every turn, add people's recombinant il-2 (Takeda Pharmaceutical Company, Ltd. product) and provide final concentration 20u/ml.As needs, active proliferative cell is divided into 2 or 3 pipes, and cultivates in the fresh culture that contains 20u/ml IL-2.

The T-cell-specific is analyzed by ELISPOT and is checked.

For setting up the T-cell clone, according to Blood, 98:1872-1881, the method described in 2001 is carried out limiting dilution to polyclone CTL.More specifically, with the positive T-cell inoculation of polyclone CD8 at 96 hole annular plates (1,3,10 cells/well), contain substratum (0.2ml) and anti--CD3 monoclonal antibody (30ng/ml) (Orth Diagnostic Inc. product) in the plate, IL-2 (50u/ml), 1 * 10 ⁵The PBMC cell and 2 * 10 of individual γ-radiation (33Gy) ⁴The B lymphoblast of the individual γ-radiation that is transformed by EBV (55Gy) (cell that obtains with 95.8-cell (JCRB9123) supernatant liquor conversion volunteer's peripheral blood B lymphocyte).

Incubation is after two weeks, the specificity of proliferating cells with the same manner of method (J.Immunol, 158:3325-3334,1997) such as Lee, kill and wound to analyze by CTL-CTL-and determine.

Ctl clone is being that 2 μ M contain same origin and are incubated overnight in derived from the 100 μ l perfect mediums of the peptide of Ep-CAM or control peptide ENV584 or 96 hole annular plates (300 cells/well) with final concentration only.

The cell killing ability of CTL cell utilizes inverted microscope to determine in next day.With these only pulse during with Ep-CAM the clone of showed cell-kill capability move to flask, and propagation as mentioned above.

6) ELISPOT of IFN-γ analyzes

Flat 96 hole MultiScreen-HA plates (Millipore Corp. product) bag with nitrocellulose filter is resisted-IFN-γ monoclonal antibody (R﹠amp with 10 μ g/ml; And under 4 ℃, be incubated overnight D systems company product).After PBS (phosphate buffered saline buffer) washing, this plate sealed 1 hour down at 37 ℃ with substratum.In each plate hole, T2-A24 cell (5 * 10 ⁴Cell) contains 0.1%FCS and 5 * 10 at 100 μ l ⁵In the RPMI1640 substratum of M beta-mercaptoethanol under the room temperature pulse with the synthetic peptide of each epi-position candidate 30 minutes.With 1 * 10 ⁵The individual CD8-positive T cell that is suspended in the substratum that contains 20%FCS is seeded in each hole.

When too many point is counted, 1 * 10 ⁴The CD8 positive T cell is used for pair cell and reacts.Whole measuring method is carried out in duplicate.

Plate 37 ℃ of following incubations 20 hours in the 5%CO2 incubator, and thorough washing is to contain the PBS of 0.05%Tween 20.Subsequently, in each hole, add multi-clone rabbit anti--IFN-gamma antibodies (Genzyme product), placed 90 minutes under the room temperature, continued to be exposed to peroxidase-bonded goat antirabbit anti-immunoglobulin G (Genzyme product) then 90 minutes.For the IFN-γ distinguished point that develops the color, in each hole, add the 0.1M sodium acetate buffer (pH 5.0) that contains 3-amino-9-ethyl carbazole (Sigma product) and 0.015% superoxol.After 40 minutes,, and make the plate drying with the water washing termination reaction.Counting greatly of diffusion counted at microscopically.

The results are shown in Fig. 1 and 2.

Fig. 1 show when the positive T-cell of the positive healthy contributors' of 5 HLA-A2402 CD8 by by a kind of pulse in 6 kinds of peptides from-ELISOT assay the result of the malicious lymphocyte series of polyclone peptide-activated born of the same parents when the deutero-dendritic cell stimulate.

As Fig. 1 finding, when the positive T-cell of the positive healthy contributors' of 5 HLA-A2402 CD8 by by a kind of pulse in 6 kinds of peptides from-when the deutero-dendritic cell stimulate, the T-clone that obtains from 4 contributors by with shown the main IFN-of generation γ point by the T2-A24 cell incubation of Ep173 pulse.

CTL system experience stimulates available from No. 3 contributors' Ep250, by with the Ep250 incubation, specificity is produced IFN-γ point.

Fig. 2 shows polyclone that the Ep-CAM peptide is special and the tetramer coloration result of mono-clonal CTL.

Fig. 2 A shows, stimulates four times polyclone CD8 positive T cell anti--CD8 antibody of FITC-mark with Ep173, contains the HLA-A24 tetramer of the PE-mark of Ep173, or control peptide ENV584 dyeing, and passes through flow cytometry analysis.The ratio of tetramer positive cell in the positive T-cell of total CD8 is with the percentages show of upper right quarter.

Shown in Fig. 2 A, in most of control peptide ENV584, do not observe IFN-γ point and form.After stimulating for four times, from CTL system that No. 4 contributors set up with the HLA-A24/Ep173 tetramer but not with HLA-A24/ENV584 tetramer specific stain (the positive T-cell of total relatively CD8,37.2%: 0.06%, Fig. 2 A).

Fig. 2 B shows, the ctl clone (C27) that Ep173-is special, dyeing in the same way as described above.The ratio of tetramer positive cell in the positive T-cell of CD8 is with the percentages show of upper right quarter.

Shown in Fig. 2 B, the intensity of tetramer positive cell is equal to or is higher than tetramer negative cells 2-to 3-doubly on logarithm.The inventor sets up T cell clone C27 from the limiting dilution substratum of No. 4 contributors' Ep173 specific polyclonal CTL system.

Utilize tetrameric this studies show that, the special CD8 positive T cell of polyclone and mono-clonal Ep173-all has the high-affinity cell receptor that is specific to the HLA-A2402/Ep173 complex body.

Therefore, the special ctl clone of Ep173-is set up from this CTL system.

Embodiment 2

The character of the ctl clone that peptide Ep173 is special, part 1

7) CTL analyzes

The inventor checks further whether C27 discerns the spontaneous peptide of presenting on tumor cell surface under the HLA-A24 situation.

The cell of Cr-mark before adding C27 be specific to total HLA I type, the monoclonal antibody incubation of any among HLA-A24 or the HLA-A2, and be 10 to carry out CTL and analyze with the ratio of effector and target.

Target cell (PC9 cell) in 100 μ l substratum under 37 ℃ with cadmium ( ⁵¹Cr) mark is 1 hour.In some experiment, in order to specify HLA restriction, before adding the effector cell 30 minutes, add the blocking antibody W6/32 (anti--HLA I type) of predetermined amount to each hole, MA2.1 (anti--HLA-A2), and A11.1 (anti--HLA-A24).

Plate is 37 ℃ of following incubations 4 hours, and utilizes γ-calculating instrument that supernatant liquor is counted.

⁵¹The calculation formula that Cr discharges % is: 100 * (experiment release-spontaneous release)/(maximum release-spontaneous release)

The results are shown in Fig. 3 and 5.

Fig. 3 A shows, is 1 o'clock at the ratio of effector and target, by ⁵¹Cr discharges and analyzes definite anti-T2-A24 of C27 (Ep173-specific CTL clone) at the peptide Ep173 of each prescribed concentration and the cellular toxicity at control peptide EBV-LMP419 place.

As a result, the special ctl clone (C27) of Ep173-confirms at the cellular toxicity of pulse with the T2-A24 cell of the Ep173 of low peptide concentration 100pM, and this cellular toxicity utilizes EBV-LMP419 (control peptide) not to be observed.

Fig. 5 and table 2 show the evaluation result of C27 at the cellular toxicity of multiple different carcinoma clone.

Table 2

	Origin	Ep-CAM RT-PCR	HLA-A24(MFI ^a)
	Origin	Ep-CAM RT-PCR	HLA-A24(MFI ^a)	Lung cancer			+(87.16)
Lu99	Large cell carcinoma	+	+(43.53)	Lung cancer			+(87.16)
Lu99	Large cell carcinoma	+	+(43.53)	PC9	Gland cancer	++	+(73.67)
11-18	Gland cancer	-	+(96.71)	PC9	Gland cancer	++	+(73.67)
11-18	Gland cancer	-	+(96.71)	LC99A	Large cell carcinoma	+	-(3.67)
LC65A	Small cell carcinoma	+	+(70.56)	LC99A	Large cell carcinoma	+	-(3.67)
LC65A	Small cell carcinoma	+	+(70.56)	LC-1/sq	Squamous cell carcinoma	+	-(3.21)
A549	Gland cancer	-	+(109.73)	LC-1/sq	Squamous cell carcinoma	+	-(3.21)
A549	Gland cancer	-	+(109.73)	A549-A24	Gland cancer	N.D. ^b	-(2.65)
QG56	Squamous cell carcinoma	+	+(84.12)	A549-A24	Gland cancer	N.D. ^b	-(2.65)
QG56	Squamous cell carcinoma	+	+(84.12)	QG56-A24	Squamous cell carcinoma	N.D.
Cancer of the stomach				QG56-A24	Squamous cell carcinoma	N.D.
Cancer of the stomach				MKN28	Gland cancer	+	-(7.67)
MKN45	Gland cancer	+	+(47.99)	MKN28	Gland cancer	+	-(7.67)
MKN45	Gland cancer	+	+(47.99)	Colorectal carcinoma
COLO320DM	Gland cancer	-	+(35.35)	Colorectal carcinoma
COLO320DM	Gland cancer	-	+(35.35)	Other cancers
HSC-2	Oral squamous cell carcinoma	±	+(34.40)	Other cancers
HSC-2	Oral squamous cell carcinoma	±	+(34.40)	K562	Leukemia	±	-(5.23)
T2-A24	B * T hybrid cell	-	+(197.75)	K562	Leukemia	±	-(5.23)

Remarks:

^a: the anti-mouse IgG antibody F (ab ') that utilizes HLA-A24mAb and FITC mark ₂Fragment by immunofluorescence, is estimated average fluorescent strength.

^b: do not do

Fig. 2 shows that C27 (ctl clone that Ep173-is special) is a evaluation result as the cellular toxicity of target cell to 8 kinds of HLA-A24 positive cancer cells system and HLA-A24 negative cells.

Ep-CAM expresses in all cells system, and removing lung adenocarcinoma cell is 11-18, beyond COLO320DM and the A549.HLA-A2402 gene such as retrovirus are transformed to A549 and QG56, and the transformant of called after A549-A24 and QG56-A24 is also as target cell.By ⁵¹Cr discharges analysis, at each effector: the ratio of target (40: 1,20: 1,10: 1,5: 1) locate, describe cellular toxicity in detail.K562 is the typical cells system that is sensitive to natural killer cell.

Shown in Fig. 5 and table 2, C27 applies toxicity effectively to lung cancer cell line PC9, LU99, LC-1/sq and LC99A; Oral cavity squamous cancerous cell line HSC-2; The stomach cancer cell of not only having expressed HLA-A24 but also having expressed Ep-CAM is MKN45.Yet C27 does not show at the positive Ep-CAM negative cells system of HLA-A24 (A549-A24 and COLO320DM), or HLA-A24 is negative and the Ep-CAM positive cell line, or Ep-CAM negative cells system (OG56, A549, toxicity MKN28).When the HLA-A2402 gene imported the negative QG56 clone of HLA-A24 (QG56-A24), C27 killed and wounded target cell.Cytotoxicity to K562 is low.

These data show, the special CTL of Ep173-kills and wounds the tumour cell of not only expressing HLA-A24 but also expressing Ep-CAM.

Fig. 3 B shows the restraining effect of the cellular toxicity of ctl clone (C27) the anti-HLA-A2402 positive cell line (PC9) that anti--HLA-A24 monoclonal antibody is special to Ep173-.

C27 is specific to the monoclonal antibody sealing of HLA-A24 or I type molecule (W6/32) to the cellular toxicity of lung cancer cell line (the positive Ep-CAM positive lung cancer of HLA-A24 clone) PC9, but not by anti--HLA-A2 monoclonal antibody sealing.

8) cold target inhibition analysis

Carry out cold target inhibition analysis according to methods such as Arai (Blood, 97:2903-2907,2001).

T2-A24 cell and final concentration are 1 * 10 ⁵The epitope peptide Ep173 of M or EBV-LMP419 incubation 1 hour.

After washing for several times, gains and 2 * 10 ⁴Effector cell's incubation 1 hour is 40: 1 with T2-A24 cell and the target cell ratio that generates the peptide pulse, 20: 1, and 10: 1 and 5: 1.2 * 10 ³Individual ⁵¹The PC9 clone of Cr-mark is added in each hole.

The definite of cellular toxicity as above states described in the 7th the relevant CTL analysis.

Fig. 3 C shows cold target inhibition analysis result.

In Fig. 3 C, the X-coordinate indicating impulse is with the T2-A24 cell (cold) of Ep173 (●) or contrast EBV-LMP419 (■) peptide and the number ratio of the PC9 cell (heat) of cadmium mark.Ordinate zou is represented the cellular toxicity of C27 at PC9 with per-cent.Open circles shows the cellular toxicity that does not have cold target.

As a result, the specificity that is subjected to the T2-A24 cell of Ep173-pulse at the cellular toxicity of the C27-of PC9 mediation suppresses, but is not subjected to the inhibition of irrelevant peptide.

Therefore, at the cellular toxicity of the C27-of PC9 mediation by the cold target cell sealing of anti--HLA-A24 monoclonal antibody or Ep173-pulse.So show, ctl clone has good specificity to the spontaneous Ep173 that presents on tumor cell surface.

Embodiment 3

The character of the ctl clone that peptide Ep173 is special, part 2

9)RT-PCR

Utilize GenElute mRNA Miniprep test kit (Sigma product), the whole RNA of extracting from the clone of cultivating.The Oligonucleolide primers of gene specific is synthetic by Proligo (ProligoJapan product), is used for the evaluation that Ep-CAM mRNA expresses.The RT-PCR the primer is as follows:

Forward primer: ATGGCGCCCCCGCAGGTCCT (SEQ ID NO:8)

Reverse primer: TTATGCATTGAGTTCCCTATGCATCTCACC (SEQ IDNO:9)

Utilize thermal cycler (Perkin-Elmer product), carry out RT-PCR, the PCR product is analyzed by 1.5% gel electrophoresis and ethidium bromide colour developing.

PCR adopted 1 round-robin 94 ℃, 5 minutes, 30 round-robin 94 ℃, 30 seconds, and 58 ℃, 30 seconds and 72 ℃, 1 minute, and 1 round-robin 72 ℃, 7 minutes.

10) western blot analysis

Step according to (J.Immunol, 165:768-778,2000) such as Schwartz that modification is arranged is slightly carried out western blot analysis.More specifically, cytolysis in damping fluid (50mM Tris/ hydrochloric acid, pH 7.5,5mM magnesium chloride, 1mM EDTA, 0.5%triton X-100,10 μ M leupeptins, 2.8 μ M pepstatins and 0.85mM phenylmethylsulfonyl fluoride) 4 ℃ following 30 minutes.In order to determine protein concentration, dissolved cell conditioned medium liquid is in the place's quantitative assay of 280/260nm wavelength.The albumen of 130 μ g further is added into 12%SDS-PAGE.Albumen, and is spent the night with the PBS sealing that contains 100% lower fat dried milk and 0.1%Tween 20 at 4 ℃ on Immobilon-P film (Millipore product) by trace.

Utilize the special monoclonal antibody of Ep-CAM (LabVision product) to retrieve, and utilize peroxidase-bonded goat anti-mouse IgG (Zymed product) to detect.

Utilize ECL western blotting detection system (Amersham bio-science company product) to make the albumen colour developing.

Fig. 4 shows that by RT-PCR and western blot analysis, cancerous cell line is expressed the check result of Ep-CAM.

There are 12 kinds (80%) obviously to express Ep-CAM in 15 kinds of cancerous cell lines.The expression utilization of HLA-A24 is anti--and the HLA-A24 monoclonal antibody checks by the indirect immunofluorescence test.In 15 kinds of cancerous cell lines checking, there are 10 kinds of clones to show that positive HLA-A24 expresses.

Shown in the foregoing description, the inventor has obtained the epi-position of the New Type of HLA-A2402-restriction derived from Ep-CAM.At least epitope peptide Ep173 (RYQLDPKFI; SEQ IDNO:1) can induce CTL (comprising the high-affinity T-cell receptors that is specific to HLA-A2402/ peptide complex body), thus, it is expected utilizing this peptide immunotherapy.

Industrial applicibility

Peptide of the present invention can be suitable the cancer vaccine as the people's cancer that contains HLA-A2402 of wide region.

Sequence table

＜110〉Otsuka Pharmaceutical Co., Ltd (Otsuka Pharmaceutical Co., Ltd.)

Like county magistrate (Aichi Prefecture)

＜120〉epi-position/peptide and application (the epitope peptide thereof that discerns by the special CTL of the Ep-CAM-of HLA-A2402-restriction

which is recognized by HLA-A2402-restricted and Ep-CAM-specific CTL)

<130>SCT063152-47

<150>JP 2004-11752

<151>2004-01-20

<160>11

<170>PatentIn version 3.1

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<223>Ep-CAM epitope peptide candidate being capable of inducing

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Claims

1. each peptide of following (1)-(4):

2. the peptide of following (1) or (2):

3. comprise the cancer vaccine of the peptide of claim 1 or 2 as activeconstituents.

4. the cancer vaccine of claim 3, wherein cancer is an epithelial cancer.

5. claim 3 or 4 cancer vaccine, wherein cancer is selected from large bowel cancer, lung cancer, mammary cancer, cancer of the stomach, oral carcinoma, carcinoma of the pancreas, esophagus cancer, nasopharyngeal carcinoma, uterus carcinoma, prostate cancer, and carcinoma of gallbladder.

6. each cancer vaccine of claim 3-5, it is used for the treatment of and has the people of HLA-A2402 as human leucocyte antigen.

7. comprise the born of the same parents poison T lymphocyte inductor of the peptide of claim 1 or 2 as activeconstituents.

8. the born of the same parents of claim 7 poison T lymphocyte inductor, it is used for the treatment of and has the people of HLA-A2402 as human leucocyte antigen.

9. each polynucleotide of following (5)-(8):

10. epitheliomatous gene therapy medicament, the polynucleotide that comprise claim 9 are as activeconstituents.

11. recombinant vectors comprises the polynucleotide of claim 9.

12. transformant wherein imports the recombinant vectors of claim 11.

13. the preparation method of the peptide of claim 1 or 2 comprises the transformant of cultivating claim 12, and the step of collecting the peptide of claim 1 or 2 from culture.

14. antigen presenting cell, its pulse is with the peptide of claim 1 or 2.

15. cancer vaccine, the antigen presenting cell that comprises claim 14 is as activeconstituents.

16. the cancer vaccine of claim 15, wherein cancer is an epithelial cancer.

17. the cancer vaccine of claim 15 or 16, wherein cancer is selected from large bowel cancer, lung cancer, mammary cancer, cancer of the stomach, oral carcinoma, carcinoma of the pancreas, esophagus cancer, nasopharyngeal carcinoma, uterus carcinoma, prostate cancer, and carcinoma of gallbladder.

18. each cancer vaccine of claim 15-17, it is used for the treatment of and has the people of HLA-A2402 as human leucocyte antigen.

19. born of the same parents' poison T lymphocyte inductor, the antigen presenting cell that comprises claim 14 is as activeconstituents.

20. the born of the same parents of claim 19 poison T lymphocyte inductor, it is used for the treatment of and has the people of HLA-A2402 as human leucocyte antigen.

21. the major histocompatibility antigen complex body comprises the peptide of major histocompatibility antigen and claim 1 or 2, perhaps is present in the tumour antigen epitope peptide on the antigen presenting cell of claim 14.

22. the major histocompatibility antigen complex body of claim 21 comprises the HLA-A2402 molecule, the peptide of B2M and claim 1 or 2 perhaps is present in the tumour antigen epitope peptide on the antigen presenting cell of claim 14.

23. cancer vaccine, the major histocompatibility antigen complex body that comprises claim 21 or 22 is as activeconstituents.

24. the cancer vaccine of claim 23, wherein cancer is an epithelial cancer.

25. the cancer vaccine of claim 23 or 24, wherein cancer is selected from large bowel cancer, lung cancer, mammary cancer, cancer of the stomach, oral carcinoma, carcinoma of the pancreas, esophagus cancer, nasopharyngeal carcinoma, uterus carcinoma, prostate cancer, and carcinoma of gallbladder.

26. each cancer vaccine of claim 23-25, it is used for the treatment of and has the people of HLA-A2402 as human leucocyte antigen.

27. born of the same parents' poison T lymphocyte inductor, the major histocompatibility antigen complex body that comprises claim 21 or 22 is as activeconstituents.

28. the born of the same parents of claim 27 poison T lymphocyte inductor, it is used for the treatment of and has the people of HLA-A2402 as human leucocyte antigen.

29. the major histocompatibility antigen complex body tetramer comprises the peptide of major histocompatibility antigen and claim 1 or 2, perhaps is present in the tumour antigen epitope peptide on the antigen presenting cell of claim 14.

30. utilize one or more following (a)-(d) materials, by stimulating born of the same parents' poison T lymphocyte of peripheral blood lymphocyte acquisition:

(a) claim 1 or 2 peptide;

(b) antigen presenting cell of claim 14;

(c) claim 21 or 22 major histocompatibility antigen complex body;

(d) the major histocompatibility antigen complex body tetramer of claim 29.

31. the born of the same parents of claim 30 poison T lymphocyte, it obtains through the following steps: one or more (a)-(d) materials that utilize claim 30 to limit, by stimulating peripheral blood lymphocyte, between major histocompatibility antigen complex body and/or its tetramer and born of the same parents' poison T lymphocyte, form complex body, and from complex body, separate born of the same parents' poison T lymphocyte.

32. the passive immunotherapy medicine, the born of the same parents' poison T lymphocyte that comprises claim 30 or 31 is as activeconstituents.

33. the passive immunotherapy medicine of claim 32, wherein cancer is an epithelial cancer.

34. the passive immunotherapy medicine of claim 32 or 33, wherein cancer is selected from large bowel cancer, lung cancer, mammary cancer, cancer of the stomach, oral carcinoma, carcinoma of the pancreas, esophagus cancer, nasopharyngeal carcinoma, uterus carcinoma, prostate cancer, and carcinoma of gallbladder.

35. each passive immunotherapy medicine of claim 32-34, it is used for the treatment of and has the people of HLA-A2402 as human leucocyte antigen.

36. the lymphocytic method of born of the same parents poison T that quantitatively HLA-A2402-limits in the peripheral blood, it comprises the following steps:

Make one or more following (a)-(d) materials act on peripheral blood:

(a) claim 1 or 2 peptide;

(b) antigen presenting cell of claim 14;

(c) claim 21 or 22 major histocompatibility antigen complex body;

(d) the major histocompatibility antigen complex body tetramer of claim 29, and

37. cancer therapy and/or improvement method comprise to having the people of HLA-A2402 as human leucocyte antigen and use one or more following (a)-(d) materials:

(a) claim 1 or 2 peptide;

(b) antigen presenting cell of claim 14;

(c) claim 21 or 22 major histocompatibility antigen complex body;

(d) the major histocompatibility antigen complex body tetramer of claim 29.

38. cancer therapy and/or improvement method comprise the following steps:

Adopt one or more following (a)-(d) materials to cultivate the monocyte part:

(a) claim 1 or 2 peptide;

(b) antigen presenting cell of claim 14;

(c) claim 21 or 22 major histocompatibility antigen complex body;

(d) the major histocompatibility antigen complex body tetramer of claim 29, and

Wherein born of the same parents poison T lymphocyte is induced and/or the activated monocyte partly is back to blood samples of patients.

39. induce the lymphocytic method of born of the same parents poison T, comprise to having the people of HLA-A2402 and use one or more following (a)-(d) materials as human leucocyte antigen:

(a) claim 1 or 2 peptide;

(b) antigen presenting cell of claim 14;

(c) claim 21 or 22 major histocompatibility antigen complex body;

(d) the major histocompatibility antigen complex body tetramer of claim 29.

40. cancer therapy or improvement method comprise to having the people of HLA-A2402 as human leucocyte antigen and use born of the same parents' poison T lymphocyte of claim 30 or 31.

41. the major histocompatibility antigen complex body tetramer of claim 21, wherein the tetramer is for comprising the HLA-A2402 molecule, the peptide of B2M and claim 1 or 2 perhaps is present in the complex body of the tumour antigen epitope peptide on the antigen presenting cell of claim 14.

42. cancer vaccine, the major histocompatibility antigen complex body tetramer that comprises claim 41 is as activeconstituents.

43. the cancer vaccine of claim 42, wherein cancer is an epithelial cancer.

44. the cancer vaccine of claim 42 or 43, wherein cancer is selected from large bowel cancer, lung cancer, mammary cancer, cancer of the stomach, oral carcinoma, carcinoma of the pancreas, esophagus cancer, nasopharyngeal carcinoma, uterus carcinoma, prostate cancer, and carcinoma of gallbladder.

45. each cancer vaccine of claim 42-44, it is used for the treatment of and has the people of HLA-A2402 as human leucocyte antigen.

46. born of the same parents poison T lymphocyte inductor comprises the major histocompatibility antigen complex body tetramer of claim 41.

47. the born of the same parents of claim 46 poison T lymphocyte inductor, it is used for the treatment of and has the people of HLA-A2402 as human leucocyte antigen.